Spectrotemporal organization of mouse ultrasonic vocalizations during neonatal opioid withdrawal and a dynorphin/kappa opioid receptor component in females
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Citation
Abstract
Opioid use during pregnancy can lead to neonatal opioid withdrawal syndrome (NOWS). NOWS is a collection of systemic and neurological withdrawal signs that appear 24 – 72 hours after birth due to the spontaneous cessation of opioid exposure in utero, including gastrointestinal and autonomic system dysfunction, irritability, and excessive high-pitched crying. NOWS severity is assessed using scoring systems, such as the Finnegan Neonatal Abstinence Scoring System (FNASS) and the Eat, Sleep, Console (ESC) approach. However, these methods are lengthy, complex, and based on subjective observations – which can lead to inconsistent evaluations, inappropriate treatment plans, and consequently, increased length of hospital stays and costs. NOWS treatments include pharmacological and non-pharmacological interventions, including opioid replacement therapies, breastfeeding, and rooming-in; however, there is no one standard care approach used across hospitals. Neonatal opioid exposure during the third trimester of human pregnancy is necessary for the presentation of NOWS in infants. The first two postnatal weeks in mice model neurodevelopmental events during the third trimester. Thus, we can effectively measure the effects of neonatal morphine exposure on opioid withdrawal behaviors and the neurobiological underpinnings. Importantly, we can model complex NOWS traits in mice that are difficult to study in the clinic, such as irritability and excessive crying. We used a third trimester-approximate mouse model for NOWS that is sufficient to induce withdrawal symptoms. Neonatal mouse pups receive twice-daily injections of morphine (10mg/kg or 15mg/kg, s.c.) or saline (0.9%, s.c.) from postnatal (P) day one to P14 or P15. Morphine exposure from P1 – P14/P15 was sufficient to induce withdrawal traits in mice, such as low body weight, thermal hyperalgesia, increased ultrasonic vocalizations (USV), and altered USV spectrotemporal profiles.
Neonatal mouse USVs signify distress and are emitted exclusively in isolation to promote maternal attention and rescue. Thus, we propose that USVs can model the negative affective state associated with NOWS. We identified a novel USV profile during spontaneous morphine withdrawal, as characterized by an increase in Complex 3 syllables in inbred FVB/NJ females and both sexes of outbred Carworth Farms Webster (CFW) mice. Notably, increased USVs and Complex 3 emissions were driven by morphine-withdrawn FVB/NJ females.
Pathway enrichment analysis of differentially expressed genes in the brainstem of FVB/NJ mice during spontaneous withdrawal revealed processes associated with synaptic organization and activity, extracellular matrix organization and glial cell development, which are disrupted during addiction and withdrawal. Notably, there was upregulation of Oprk1 in the brainstem, which codes for the kappa opioid receptor (κOR), and increased expression of Pdyn, which codes for the endogenous κOR ligand, dynorphin, in the midbrain. The dynorphin/κOR system is known to mediate withdrawal-induced dysphoria; thus, we aimed to identify the role of κOR activation in mediating the altered USV profile observed during neonatal morphine withdrawal. Using a κOR antagonist to block κOR activation during withdrawal, we observed a reduction in USVs in females. We also administered a κOR agonist to morphine-naïve mice and found increased USVs in both sexes on P10 and in females on P14. These observations are consistent with a female-specific component of κOR in mediating affective NOWS symptom severity.
Excessive, high-pitched crying is one of the most prominent withdrawal symptoms used to assess NOWS severity in infants. However, NOWS assessment is based on subjective scoring systems and can vary between observers and across hospitals, leading to inappropriate diagnoses and treatment interventions. Thus, non-subjective or quantitative measures of NOWS cries may more effectively assess NOWS severity and guide optimal treatment options. We observed changes in USV frequency (pitch), length, and power (volume) during spontaneous morphine withdrawal in four inbred substrains of FVB/N mice akin to altered cries in human infants. These observations suggest that there may be additional acoustic features associated with the severity of neonatal opioid withdrawal that could be objectively measured in humans and inform treatment options.
These results provide insight into the neurobiological mechanisms mediating affective NOWS symptom severity in females. We identified USV features that are unique to the dysphoric state associated with morphine withdrawal, which may serve as better predictors for NOWS symptom severity compared to current observation-based assessment tools.
Description
2025
License
Attribution-NoDerivatives 4.0 International